Compositions useful in the treatment of prostatic cancer and related conditions are described in U.S. Pat. Nos. 5,599,686 and 5,866,679; and U.S. patent application Ser. No. 08/950,805, filed 14 Oct. 1997 (corresponding to International Patent Publication No. WO 98/18493) entitled Conjugates Useful in the Treatment of Prostate Cancer. Said compositions, which may be termed PSA conjugates, comprise chemical conjugates comprising known cytotoxic agents and oligopeptides having amino acid sequences that are selectively proteolytically cleaved by free prostate specific antigen and, with respect to Ser. No. 08/950,805 (corresponding to U.S. Pat. No. 5,948,750, issued on 7 Sep. 1999), that include a cyclic amino acid having a hydrophilic substituent. The oligopeptide moieties are selected from oligomers that are selectively recognised by free prostate specific antigen (PSA) and are capable of being proteolytically cleaved by the enzymatic activity thereof.
Ideally, the cytotoxic activity of the cytotoxic agent is greatly reduced or absent when the intact oligopeptide containing the PSA proteolytic cleavage site is bonded directly, or through a chemical linker, to the cytotoxic agent. Also ideally, the cytotoxic activity of the cytotoxic agent increases significantly, or is restored completely, upon proteolytic cleavage of the attached oligopeptide at the cleavage site. Preferably, the N-terminus of the oligopeptide is protected by a hydrophilic blocking group, of which glutaric acid and succinic acid are preferred examples. Such protected oligopeptides may be illustrated by the following structure:Protecting group—AA1-AA2-AA3-AA4-AA5-AA6-AA7 wherein AA1, AA2, AA3, AA4, AA5, AA6 and AA7 are independently selected from a natural and unnatural amino acid. It is understood that protected oligopeptides having greater or fewer amino acid residues (from 5 to 10 amino acids) may alternatively be incorporated in the PSA conjugate.
Among the preferred N-terminus protecting groups that are incorporated onto a PSA conjugate are the dicarboxylic acid alkanes, such as succinyl, glutaryl and the like. Therefore a preferred protected oligopeptide may be illustrated by the formula:

To ensure selective attachment of the cytotoxic agent via the AA7 residue, the free carboxylic acid group of the protecting group must be blocked. A suitable blocking group for this purpose is the 9-fluorenylmethyl ester (Fm), since it is readily removed under mild conditions (20% piperidine) at the end of the process. Thus, key intermediates in the synthesis of the desired PSA conjugates are compounds of formula A:

In several of the specific examples disclosed in WO 98/18493, the conjugate comprises an oligopeptide having the amino acid sequence:4-Hydroxyproline-alanine-serine-AA4-AA5-AA6-AA7 and the cytotoxic agent is attached to the C-terminus (i.e. via the carboxyl group of the AA7 residue). Thus, in a preferred preparative process, the desired cytotoxic agent is attached to the AA7 residue of a peptide analogue of Formula B:
where Fm represents 9-fluorenylmethyl and r is 2 or 3.
As disclosed in the above-referenced patent application, such compounds may be prepared by a linear strategy involving conventional techniques of solid-phase peptide synthesis. However, such methods are best suited to laboratory-scale synthesis, rather than factory-scale preparations. Furthermore, the solid-phase process requires the use of anhydrous HF, which necessitates special handling techniques and precautions.
An alternative strategy, more amenable to scale-up, involves the preparation of a tripeptide analogue of Formula (C):
and in particular the intermediate compound of the Formula (C-1)
where Fm and r are as previously defined, followed by coupling of the protected tripeptide (C) to the appropriate tetrapeptide.
Compounds of Formula (C-1) therefore represent important synthetic targets. Although a conventional solution-phase strategy, starting with serine, may be employed for the synthesis of such compounds, the results are disappointing. In particular, it is necessary to protect both the hydroxyl group and the carboxylic acid group of serine (e.g. as the benzyl ether and p-nitrobenzyl ester, respectively) during the assembly of the peptide chain and the introduction of the Fm-blocked glutaryl or succinyl group. Attempts to remove these protecting groups invariably lead to partial cleavage of the Fm blocking group, with consequent reductions in yield and/or purity of the desired products.
Thus, there is a continuing need for a convenient, clean, high-yield process for the synthesis of peptidyl intermediate compounds useful in the synthesis of PSA conjugates, in particular the intermediate compounds of Formula (C) and the precursor compounds of the Formula (A), suitable for use on an industrial scale.